A major hypothesis of CNS injury is that the increased extracellular excitatory amino acid (EAA) levels which occur after such injuries cause increased intra-neuronal calcium levels due to excessive activation of glutamate receptors, leading to neuronal damage and death. This scheme is supported by several lines of evidence such as, that treatment with glutamate receptor blockers or inhibition of the increased EAA levels in vivo are protective. In sharp contrast to the considerable body of work on the effects of the increased EAA levels, the cellular sources and mechanisms of the increased EAR have been little-studied. Both increased release and decreased uptake would contribute to increased EAAs. Surprisingly, in recent microdialysis studies, much of the increased EAA levels seen in animal models of ischemia has been found to be Ca2+ insensitive, suggesting that exocytotic release from nerve terminals is not a major contributor to the increased EAA levels, and that other mechanisms and sources must be considered. Extracellular K+ increases markedly in ischemia, and two Ca2+-independent release mechanisms for EAAs have been identified in in vitro which are stimulated by raised K+. These are reversal of the glutamate transporter seen in synaptosomes and cultured neurons and astrocytes, and swelling- induced release of EAAs seen in astrocytes. We will study primary astrocyte cultures and synaptosomes derived from the hippocampus and cultured cerebellar granule cells for our in vitro studies, as there are no hippocampal neuronal cultures that provide sufficient quantities of cells for transport studies. By confirming the transporter forms seen in the different in vitro preparations we will see if they have different reversal characteristics. Another major contributor to the increased EAA levels would be inhibition of uptake and astrocytes have very active and astrocyte-specific EAA uptake systems. We will study inhibition of EAA uptake by increased medium K+ in astrocytes in vitro. Using the data obtained on the different systems in vitro, we will determine their contributions to the increased levels of EAAs in vivo by microdialysis experiments in the rat hippocampus with reversible global ischemia. In addition we will produce localized depletion of astrocytes with gliotoxins and neurons by kainic acid, and determine the influence of these procedures on the ischemia-induced release of EAAs. Understanding the different sources and mechanisms of EAA release or impaired uptake and their respective contributions to the elevated EAA levels in ischemia and other pathological states is vital to interpretation of current therapies for such states and devising new ones.